PROJECT SUMMARY/ABSTRACT Despite intense research, we lack even the most basic understanding of how structural and functional changes in small vessel diseases of the brain (SVD) are linked to tissue oxygenation, whether this results in tissue hypoxia (O2 supply-demand mismatch), and how different microvascular segments contribute to this mismatch in different brain areas. Leveraging our cutting-edge optical imaging tools for absolute pO2, blood flow, Ca2+ signaling, and microvascular morphology, and a clinically relevant CADASIL mouse model, we propose to examine for the first time a causal link between microvascular dysfunction and O2 supply-demand mismatch. CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is the most common monogenic inherited form of SVD leading to dementia, caused by mutations in Notch3. Transgenic models of CADASIL recapitulate many clinical and histopathological hallmarks of the disease, including early signs of SVD such as impaired CBF and functional hyperemic responses, and functional and structural abnormalities in both arterioles and capillaries. We will quantify absolute intravascular and tissue pO2, metabolic rate of O2 (CMRO2), CBF and capillary red blood cell flux, and microvascular morphology, at rest and during functional brain activation, in both gray and white matter, longitudinally over months, in unanesthetized CADASIL transgenic mice. Aim 1 will test whether CADASIL causes age-dependent O2 supply-demand mismatch, manifested in islets of tissue hypoxia at rest or during functional activation. Combining two-photon microscopy and optical coherence tomography (OCT), we will test whether CADASIL leads to abnormal capillary red blood cell flux and increased capillary transit time heterogeneity causing O2 supply-demand mismatch even before a reduction in absolute CBF becomes manifest. Finally, we will examine whether O2 supply-demand mismatch can be corrected by genetic, pharmacological, and immunological manipulations. Aim 2 will test whether CADASIL causes Ca2+ dysregulation in vascular smooth muscle and pericytes and whether it can be corrected by genetic, pharmacological and immunological manipulations. Aim 3 will integrate the measurements from Aims 1 and 2 and construct a numerical model of oxygen advection and diffusion based on measured microvascular morphology, reactivity, perfusion, and oxygenation. The model will relate structural and functional microvascular changes to tissue O2 supply-demand mismatch, quantify the contribution and predict the limits of the arteriolar and capillary compartments to support tissue oxygenation below which O2 supply-demand mismatch develops. The model will thus shed light on all other SVDs (e.g. hypertension, amyloid). Altogether, this proposal aims to fill significant gaps in our understanding of the mechanisms of microvascular dysfunction in CADASIL, and will inform the vascular mechanisms of progressive neurodegeneration and cognitive impairment in more common forms of SVD as well as in Alzheimer?s disease and related dementias.